The present invention relates generally to apoptosis, or programmed cell death, and more particularly, to a novel aspartate-specific cysteine protease that can be used to modulate apoptosis for the therapeutic treatment of human diseases.
Apoptosis is a normal physiological process of cell death that plays a critical role in regulating tissue homeostasis by ensuring that the rate of new cell accumulation produced by cell division is offset by a commensurate rate of cell loss due to cell death. It has now become clear that disturbances in apoptosis, also referred to as physiological cell death or programmed cell death, which prevent or delay normal cell turnover, can be just as important to the pathogenesis of diseases as known abnormalities in the regulation of proliferation and the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death.
The stimuli that regulate the function of these apoptotic gene products include both extracellular and intracellular signals. Either the presence or the removal of a particular stimulus can be sufficient to evoke a positive or negative apoptotic signal. For example, physiological stimuli that prevent or inhibit apoptosis include, for example, growth factors, extracellular matrix, CD40 ligand, viral gene products, neutral amino acids, zinc, estrogen and androgens. In contrast, stimuli that promote apoptosis include growth factors such as tumor necrosis factor (TNF), Fas, and transforming growth factor xcex2 (TGFxcex2). Other stimuli that promote apoptosis include, for example, neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment, intracellular calcium and glucocorticoids. Other stimuli, including those of environmental and pathogenetic origins, can either induce or inhibit programmed cell death. Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately feed into a cell death pathway that is evolutionarily conserved between humans and invertebrates.
Several gene products that modulate the apoptotic process have now been identified. Although these products can be generally separated into two basic categories, gene products from each category can function to either inhibit or induce programmed cell death. One family of gene products is related to the protein Bcl-2, which inhibits apoptosis when overexpressed in cells. Other members of this gene family include, for example, Bax, Bak, Bcl-xL, Bcl-xS, and Bad. While some of these proteins can prevent apoptosis, others augment apoptosis, for example, Bcl-xS and Bak, respectively.
A second family of gene products, the aspartate-specific cysteine proteases (ASCPs), are genetically related to the ced-3 gene product, which was initially shown to be required for programmed cell death in the roundworm, C. elegans. The ASCP family of proteases includes human ICE (interleukin-1-xcex2 converting enzyme), ICH-1L, ICH-1S, CPP32, Mch2, Mch3, Mch4, Mch5, ICH-2 and ICErel-III. Among the common features of these gene products are that 1) they are cysteine proteases with specificity for substrate cleavage at Asp-x bonds, 2) they share a relatively conserved pentapeptide sequence, QACRG (SEQ ID NO:79) or QACQG (SEQ ID NO:80), within the active site and 3) they are synthesized as proenzymes that require proteolytic cleavage at specific aspartate residues for activation of protease activity. In the case of ICE, cleavage of the proenzyme produces two polypeptide protease subunits of approximately 20 kDa, known as p20, and 10 kDa, known as p10, that combine non-covalently to form a tetramer comprising two p20:p10 heterodimers. Although these proteases induce cell death when expressed in cells, several alternative structural forms, such as ICExcex4, ICExcex5, ICH-1S and Mch2xcex2, actually function to inhibit apoptosis.
In addition to the Bcl-2 and ASCP gene families that play a role in apoptosis in mammalian cells, it has become increasingly apparent that other gene products that are important in mammalian cell death have yet to be identified. For example, in addition to Ced-3, another C. elegans gene known as Ced-4 is also required for programmed cell death in C. elegans. However, mammalian homologs of Ced-4 remain elusive and have not yet been identified. Further, it is ambiguous whether other genes belong to either of the above two apoptotic gene families or what role they may play in the programmed cell death pathway. Finally, it is unclear what physiological control mechanisms regulate programmed cell death or how the cell death pathways interact with other physiological processes within the organism. For example, it has recently been suggested that cytotoxic T-lymphocytes mediate their destructive function by inducing apoptosis in their target cells.
Apoptosis maintains tissue homeostasis in a range of physiological processes such as embryonic development, immune cell regulation and normal cellular turnover. Therefore, the dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes such as that occurring with many autoimmune diseases. Inappropriate loss of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments that are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can change the natural progression of many of these diseases.
Thus, there exists a need to identify new apoptotic genes and their gene products to modulate apoptosis for the therapeutic treatment of human diseases. The present invention satisfies this need and provides related advantages as well.
The invention provides an isolated gene encoding Mch6 as well as functional fragments thereof. Also provided are isolated nucleic acid sequences encoding Mch6 or functional fragments thereof. The gene or nucleic acid sequences can be single or double stranded nucleic acids corresponding to coding or non-coding strands of the Mch6 nucleotide sequences. The invention further provides an isolated Mch6 polypeptide and isolated large and small subunits of the Mch6 polypeptide, including functional fragments thereof.